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Systemic Dissemination of Viral Vectors During Intratumoral Injection

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Systemic Dissemination of Viral Vectors During Intratumoral Injection Molecular Cancer Therapeutics 1233 Systemic dissemination of viral vectors during intratumoral injection Yong Wang,1 Jim Kang Hu,2 Ava Krol,1 therapy (1, 2). However, the efficacy of gene therapy is Yong-Ping Li,2 Chuan-Yuan Li,2 and Fan Yuan1 still limited by the delivery of therapeutic genes into 1 2 target cells. Systemic delivery of viral vectors is inade- Departments of Biomedical Engineering and Radiation quate, primarily due to the poor interstitial penetration in Oncology, Duke University, Durham, NC solid tumors (3, 4) and normal tissue toxicity caused by viral vectors and/or gene products (5–15). Different approaches have been developed for reducing Abstract the toxicity in normal tissues. One is to switch to non-viral Intratumoral injection is a routine method for local viral vectors, such as cationic liposomes or polymers (16, 17). gene delivery that may improve interstitial transport of viral Non-viral vectors are less toxic and may have similar vectors in tumor tissues and reduce systemic toxicity. transfection efficiency in vitro as viral vectors. However, However, the concentration of transgene products in non-viral vectors are in general less efficient in vivo. The normal organs, such as in the liver, may still exceed normal second approach is to use tissue targeting viral vectors tissue tolerance if the products are highly toxic. The (18–23), which can be achieved through at least two elevated concentration in normal tissues is likely to be mechanisms. One is to incorporate specific molecular caused by the dissemination of viral vectors from the structures on the vector surface that can bind to unique tumor. Therefore, we investigated transgene expression in markers on the plasma membrane of cells or extracellular the liver, the serum, and a mouse mammary carcinoma matrix in tumors; another is to incorporate specific (4T1) in mice after intratumoral injection of adenoviral transcriptional promoters in viral vectors that can be vectors for mouse interleukin-12, luciferase, enhanced triggered by either endogenous factors or exogenous green fluorescence protein, or B-galactosidase. We also interventions. In all these cases, transgene expression is performed numerical simulations of virus transport in restricted in target cells or tissues. However, targeted gene tumors after intratumoral injection, based on the Krogh delivery requires identification of unique markers in cells cylinder model. Our experimental data and numerical and tissues that can capture the vectors or identification of simulations demonstrated that virus dissemination was specific transcriptional mechanisms that can control gene significant in mice and it occurred mainly during the expression. Both requirements cannot be always achieved. intratumoral injection. To reduce virus dissemination, we The two approaches mentioned above can reduce the mixed these vectors with a viscous alginate solution and toxicity in normal tissues, but they cannot solve the problem injected the mixture into the tumors. Our data showed that of poor interstitial penetration if the vectors are delivered the alginate solution could significantly reduce virus systemically. To simultaneously improve interstitial trans- dissemination while having minimal effects on transgene port and reduce normal tissue toxicity, the optimal expression in tumors and on interleukin-12-induced tumor approach is to locally inject viral vectors into tumors growth delay. These data suggest that virus dissemination (24, 25). Intratumoral injection can improve interstitial is a potential problem in local viral gene therapy of cancer transport through at least three mechanisms. One is to and that the dissemination could be significantly reduced establish a pressure gradient for enhancing convection, by the alginate solution without compromising the efficacy which is critical for delivery of macromolecules and of gene therapy. (Mol Cancer Ther. 2003;2:1233–1242) nanoparticles (3, 4). The second is to increase the pore size in tumor tissues due to pressure-induced tissue deforma- tion (26, 27). Tissue deformation will also, as the third Introduction mechanism, improve the connectedness of interstitial space (28). When injected directly into tumors, viral vectors are Discoveries in molecular and cell biology have led to a expected to infect only cells near the injection site and hence significant development in novel strategies for cancer gene cause minimal toxicity in normal tissues. However, data in the literature have shown that the normal tissue cytotoxicity has been a limiting factor for achieving an optimal dose of Received 3/27/03; revised 7/20/03; accepted 8/7/03. viral vectors in tumors (29, 30). The concentration of The costs of publication of this article were defrayed in part by the transgene products in the liver can be on the same order payment of page charges. This article must therefore be hereby of magnitude as that in tumor tissues (31, 32). marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The normal tissue toxicity is likely to be caused by Grant support: National Science Foundation (BES-9984062) and the viruses disseminated from tumors. Virus dissemination NIH (CA81512). has been suggested in previous studies (29–34), although Requests for Reprints: Fan Yuan, Department of Biomedical Engineering, the significance of this problem and mechanisms of the 136 Hudson Hall, Box 90281, Duke University, Durham, NC 27708. Phone: (919) 660-5411; Fax: (919) 684-4488. dissemination remain to be determined. There exist at E-mail: [email protected] least two possible mechanisms of dissemination: (a) direct Downloaded from mct.aacrjournals.org on September 30, 2021. © 2003 American Association for Cancer Research. 1234 Virus Dissemination in Local Gene Therapy injection of viral vectors into blood vessels that are Type Culture Collection, Manassas, VA), harvested at 48 h damaged by the injection needle; and (b) diffusion of after infecting cells, and purified through cesium chloride viral vectors into microvessels after intratumoral injection. gradient centrifugation according to a standard protocol To demonstrate virus dissemination, we injected adeno- (35). Viral vectors were stored in 10% glycerol at À80jC. viral vectors encoding either mouse interleukin-12 (IL-12), The virus suspensions were mixed with the solution of luciferase, or enhanced green fluorescence protein (EGFP) alginate (Sigma Chemical Co., MO) or PBS before intra- into tumors, measured IL-12 concentrations in the liver, tumoral injection. The final concentration of the alginate serum, and tumors, and compared qualitatively the was 4% w/w concentration. expressions of luciferase and EGFP in the liver and Measurement of Solution Viscosity tumors, respectively. In addition, we developed a math- Brookfield DV-III Rheometer (Brookfield, MA) was ematical model of virus transport for determining which used to measure the viscosity of solutions. The temper- of the two mechanisms mentioned above was dominant in ature of water bath was set at 37jC. The samples were virus dissemination. Our experimental data and numerical put into the center of the cup and preheated for 10 min simulations demonstrated that virus dissemination was a before the viscosity measurement. The measurement was potential problem in local viral gene therapy of cancer, repeated three times and the average value is reported in and that the dissemination occurred mainly during the this paper. intratumoral injection. EGFP Expression in Different Tissues Based on the mechanistic studies mentioned above, we Fifty microliters of AdCMVEGFP in PBS or alginate further developed a method for reducing the systemic solution were injected into 4T1 tumors via a 30-gauge dissemination of viral vectors. The method was based on needle. The dose of injection was either 1.0 Â 108 or 5.0 Â the biological and physical properties of alginate solu- 108 plaque-forming units (pfu)/tumor. The samples of the tions, which are biocompatible and biodegradable and are blood, the spleen, the lung, the kidney, the heart, the liver, highly viscous. The alginate solution, when mixed with and tumors were harvested at 2 days after virus injection. viral vectors before intratumoral injection, can reduce both Then, the samples were glued onto a specimen block and the rates of convection and diffusion of viral vectors. In transferred to the stage of a Vibratome (Model 3000; addition, intratumoral injection of this solution requires a Technical Products International, St. Louis, MO) main- higher pressure gradient which may cause a transient tained at 4jC. The tissues were then sectioned into compression of blood vessels in tumors. As a result, the 300-Am slices. EGFP expression in tissues was examined virus dissemination was reduced significantly when we under a confocal laser scanning microscope (LSM 510, injected into tumors with the mixture of viral vectors and Carl Zeiss, Thornwood, NY). the alginate solution rather than the free suspension of Luciferase Expression in Different Tissues viral vectors. Fifty microliters of AdCMVLUC in PBS or alginate solution were injected into 4T1 tumors via a 30-gauge 8 Materials and Methods needle. The dose of injection was 2.0 Â 10 pfu/tumor. At 2 days after virus injection, mice were anesthetized with Tu m o r Mo d e l s i.p. injection of 80 mg ketamine and 10 mg xylazine/kg 4T1 mouse mammary carcinoma cells were obtained body weight. Then, 50 Al of aqueous D-luciferin solution from Dr. Fred Miller’s lab (Michigan
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